Endo-beta-galactosidase from Escherichia freundii has been useful for structural and immunochemical analysis of cell surface antigens such as Ii, ABH, F9, and SSEA-1. Further development of endo-beta-galactosidase will provide an indispensable tool in the analysis of the role of cell-surface carbohydrate chains in differentiation and tumorigenicity. Expecting that endo-beta-galactosidase from different origins has substrate specificities different from each other, endo-beta-galactosidases found in Diplococcus pneumoniae and Bacterioides fragilis have been purified and characterized. The former enzyme is found to be unique in that the enzyme hydrolyzes the lactosaminoglycan (LAG) by stepwise endoglycosidic action. The latter was found to be similar to E.\freundii enzyme. Since antigens Ii, F9, and SSEA-1 are specifically expressed at different stages of mouse embryonal differentiation, glycoconjugate profiles of cell surfaces were analyzed by using mouse terato-carcinoma cell lines as a model system. "Nullipotent" embryonal carcinoma cell line (F9) was stimulated to differentiate into parietal endoderm in the presence of retinoic acid and dibutyryl cyclic AMP or into visceral endoderm in the presence of retinoic acid. Surface glycoconjugates of each stage of cells were analyzed as to whether each component has polylactosamino structure characterized by susceptibility to endo-beta-galactosidase. In addition, LAG was isolated from human embryonal carcinoma cell line PAl. The structure of embryonal LAG was analyzed by endo-beta-galactosidase treatment, methylation analysis, and fast atom bombardment mass spectrometry. PAl lactosaminoglycans were found to have new disialosylterminal NeuAcd2\\9NeuAcd2\\3/6Gal, which is expected to be specific to early embryo. (A)